Peptide Oligonucleotide Conjugates

ABSTRACT

Provided herein are oligonucleotides, peptides, and peptide-oligonucleotide-conjugates. Also provided herein are methods of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject oligonucleotides, peptides, and peptide-oligonucleotide-conjugates described herein.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/163,960, filed on May 19, 2015, and U.S. Provisional PatentApplication No. 62/337,536, filed on May 17, 2016. The entire contentsof these applications are herein incorporated by reference.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing incomputer readable format. The Sequence Listing is provided as a fileentitled 581432 SPT-001-2_sequence_listing_ST25.txt, created May 13,2016, which is 4,227 bytes in size. The information in the computerreadable format of the sequence listing is incorporated herein byreference in its entirety.

BACKGROUND

Antisense technology provides a means for modulating the expression ofone or more specific gene products, including alternative spliceproducts, and is uniquely useful in a number of therapeutic, diagnostic,and research applications. The principle behind antisense technology isthat an antisense compound, e.g., an oligonucleotide, which hybridizesto a target nucleic acid, modulates gene expression activities such astranscription, splicing or translation through any one of a number ofantisense mechanisms. The sequence specificity of antisense compoundsmakes them attractive as tools for target validation and genefunctionalization, as well as therapeutics to selectively modulate theexpression of genes involved in disease.

Although significant progress has been made in the field of antisensetechnology, there remains a need in the art for oligonucleotides, andpeptide-oligonucleotide-conjugates with improved antisense or antigeneperformance. Such improved antisense or antigene performance includes,at least, for example: lower toxicity, stronger affinity for DNA and RNAwithout compromising sequence selectivity, improved pharmacokinetics andtissue distribution, improved cellular delivery, and both reliable andcontrollable in vivo distribution.

SUMMARY

Provided herein are peptides, oligonucleotides, andpeptide-oligonucleotide-conjugates. Also provided herein are methods oftreating a disease in a subject in need thereof, comprisingadministering to the subject a peptide-oligonucleotide-conjugatedescribed herein.

Accordingly, in one aspect, provided herein is apeptide-oligonucleotide-conjugate of Formula I:

or a pharmaceutically acceptable salt thereof,

In one embodiment, the peptide-oligonucleotide-conjugate of Formula I isa peptide-oligonucleotide-conjugate of Formula Ia:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the peptide-oligonucleotide-conjugate of FormulaI is a peptide-oligonucleotide-conjugate of Formula Ib:

or a pharmaceutically acceptable salt thereof.

In still another embodiment, the peptide-oligonucleotide-conjugate ofFormula I is a peptide-oligonucleotide-conjugate of Formula Ic:

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the peptide-oligonucleotide-conjugate ofFormula I is a peptide-oligonucleotide-conjugate of Formula Id:

or a pharmaceutically acceptable salt thereof

In still another embodiment, the peptide-oligonucleotide-conjugate ofFormula I is a peptide-oligonucleotide-conjugate of Formula Ie:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is apeptide-oligonucleotide-conjugate of Formula IV:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is apeptide-oligonucleotide-conjugate of Formula IV:

or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is a method of treating amuscle disease, a viral infection, or a bacterial infection in a subjectin need thereof, comprising administering to the subject apeptide-oligonucleotide-conjugate of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the extent of penetration of selected oligonucleotides,peptides, and peptide-oligonucleotide-conjugates into HeLa cells basedon flow cytometry experiments.

FIG. 2A is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 5.

FIG. 2B is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 6.

FIG. 2C is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 8.

FIG. 2D is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 10.

FIG. 3A is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 5.

FIG. 3B is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 7.

FIG. 3C is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 9.

FIG. 3D is a western blot showing dystrophin levels in mouse quadricepstissue following administration of PPMO 11.

FIG. 4A shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay).

FIG. 4B shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 6.

FIG. 4C shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay).

FIG. 4D shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 6; wildtype and mdx mice were used as controls.

FIG. 5A shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 8.

FIG. 5B shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 10.

FIG. 5C shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 8; wildtype and mdx mice were used as controls.

FIG. 5D shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 10; wildtype and mdx mice were used as controls.

FIG. 6A shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay).

FIG. 6B shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 7.

FIG. 6C shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay).

FIG. 6D shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 7; wildtype and mdx mice were used as controls.

FIG. 7A shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 9.

FIG. 7B shows the quantification of dystrophin levels in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 11.

FIG. 7C shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 9; wildtype and mdx mice were used as controls.

FIG. 7D shows the quantification of Exon23 Skipping (%) in mousequadriceps tissue following administration of PPMO 5 (two replicationsof same assay) or PPMO 11; wildtype and mdx mice were used as controls.

DETAILED DESCRIPTION

Provided herein are peptides, oligonucleotides, andpeptide-oligonucleotide-conjugates. Also provided herein are methods oftreating a disease in a subject in need thereof, comprisingadministering to the subject a peptide-oligonucleotide-conjugatedescribed herein. The oligonucleotides, and thereby thepeptide-oligonucleotide-conjugates, described herein display strongeraffinity for DNA and RNA without compromising sequence selectivity,relative to native or unmodified oligonucleotides. In some embodiments,the oligonucleotides of the disclosure minimize or prevent cleavage byRNase H. In some embodiments, the antisense oligonucleotides of thedisclosure do not activate RNase H.

The peptides described herein impart to their correspondingpeptide-oligonucleotide-conjugates lower toxicity, enhance the activityof the oligonucleotide, improve pharmacokinetics and tissuedistribution, improve cellular delivery, and impart both reliable andcontrollable in vivo distribution.

Definitions

Listed below are definitions of various terms used to describe thisdisclosure. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used. Asused herein when referring to a measurable value such as an amount, atemporal duration, and the like, the term “about” is meant to encompassvariations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

The term “alkyl” refers to saturated, straight- or branched-chainhydrocarbon moieties containing, in certain embodiments, between one andsix, or one and eight carbon atoms, respectively. Examples of C₁₋₆-alkylmoieties include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties; andexamples of C₁₋₈-alkyl moieties include, but are not limited to, methyl,ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl,heptyl, and octyl moieties.

The number of carbon atoms in an alkyl substituent can be indicated bythe prefix “C_(x-y),” where x is the minimum and y is the maximum numberof carbon atoms in the substituent. Likewise, a C_(x) chain means analkyl chain containing x carbon atoms.

The term “heteroalkyl” by itself or in combination with another termmeans, unless otherwise stated, a stable straight or branched chainalkyl group consisting of the stated number of carbon atoms and one ortwo heteroatoms selected from the group consisting of O, N, and S, andwherein the nitrogen and sulfur atoms may be optionally oxidized and thenitrogen heteroatom may be optionally quaternized. The heteroatom(s) maybe placed at any position of the heteroalkyl group, including betweenthe rest of the heteroalkyl group and the fragment to which it isattached, as well as attached to the most distal carbon atom in theheteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃, —CH₂—CH₂—CH₂-0H,—CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂—CH₂—S(═O)—CH₃. Up to twoheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃, or—CH₂—CH₂—S—S—CH₃.

The term “aryl,” employed alone or in combination with other terms,means, unless otherwise stated, a carbocyclic aromatic system containingone or more rings (typically one, two, or three rings), wherein suchrings may be attached together in a pendent manner, such as a biphenyl,or may be fused, such as naphthalene. Examples of aryl groups includephenyl, anthracyl, and naphthyl. In various embodiments, examples of anaryl group may include phenyl (e.g., C₆-aryl) and biphenyl (e.g.,C₁₂-aryl). In some embodiments, aryl groups have from six to sixteencarbon atoms. In some embodiments, aryl groups have from six to twelvecarbon atoms (e.g., C₆₋₁₂-aryl). In some embodiments, aryl groups havesix carbon atoms (e.g., C₆-aryl).

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. Heteroaryl substituents may bedefined by the number of carbon atoms, e.g., C₁₋₉-heteroaryl indicatesthe number of carbon atoms contained in the heteroaryl group withoutincluding the number of heteroatoms. For example, a C₁₋₉-heteroaryl willinclude an additional one to four heteroatoms. A polycyclic heteroarylmay include one or more rings that are partially saturated. Non-limitingexamples of heteroaryls include pyridyl, pyrazinyl, pyrimidinyl(including, e.g., 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl,pyrrolyl (including, e.g., 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl,pyrazolyl (including, e.g., 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Non-limiting examples of polycyclic heterocycles and heteroaryls includeindolyl (including, e.g., 3-, 4-, 5-, 6- and 7-indolyl), indolinyl,quinolyl, tetrahydroquinolyl, isoquinolyl (including, e.g., 1- and5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl(including, e.g., 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl,1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin,1,5-naphthyridinyl, benzofuryl (including, e.g., 3-, 4-, 5-, 6- and7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl(including, e.g., 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl,benzothiazolyl (including, e.g., 2-benzothiazolyl and 5-benzothiazolyl),purinyl, benzimidazolyl (including, e.g., 2-benzimidazolyl),benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl,pyrrolizidinyl, and quinolizidinyl.

The term “protecting group” or “chemical protecting group” refers tochemical moieties that block some or all reactive moieties of a compoundand prevent such moieties from participating in chemical reactions untilthe protective group is removed, for example, those moieties listed anddescribed in T.W. Greene, P.G.M. Wuts, Protective Groups in OrganicSynthesis, 3rd ed. John Wiley & Sons (1999). It may be advantageous,where different protecting groups are employed, that each (different)protective group be removable by a different means. Protective groupsthat are cleaved under totally disparate reaction conditions allowdifferential removal of such protecting groups. For example, protectivegroups can be removed by acid, base, and hydrogenolysis. Groups such astrityl, monomethoxytrityl, dimethoxytrityl, acetal andtert-butyldimethylsilyl are acid labile and may be used to protectcarboxy and hydroxy reactive moieties in the presence of amino groupsprotected with Cbz groups, which are removable by hydrogenolysis, andFmoc groups, which are base labile. Carboxylic acid moieties may beblocked with base labile groups such as, without limitation, methyl, orethyl, and hydroxy reactive moieties may be blocked with base labilegroups such as acetyl in the presence of amines blocked with acid labilegroups such as tert-butyl carbamate or with carbamates that are bothacid and base stable but hydrolytically removable.

Carboxylic acid and hydroxyl reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups may be blocked with base labile groups such as Fmoc.A particularly useful amine protecting group for the synthesis ofcompounds of Formula (I) is the trifluoroacetamide. Carboxylic acidreactive moieties may be blocked with oxidatively-removable protectivegroups such as 2,4-dimethoxybenzyl, while coexisting amino groups may beblocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(0)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

The term “nucleobase,” “base pairing moiety,” “nucleobase-pairingmoiety,” or “base” refers to the heterocyclic ring portion of anucleoside, nucleotide, and/or morpholino subunit. Nucleobases may benaturally occurring, or may be modified or analogs of these naturallyoccurring nucleobases, e.g., one or more nitrogen atoms of thenucleobase may be independently at each occurrence replaced by carbon.Exemplary analogs include hypoxanthine (the base component of thenucleoside inosine); 2, 6-diaminopurine; 5-methyl cytosine;C5-propynyl-modified pyrimidines; 10-(9-(aminoethoxy)phenoxazinyl)(G-clamp) and the like.

Further examples of base pairing moieties include, but are not limitedto, uracil, thymine, adenine, cytosine, guanine and hypoxanthine havingtheir respective amino groups protected by acyl protecting groups,2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil,2,6-diaminopurine, azacytosine, pyrimidine analogs such aspseudoisocytosine and pseudouracil and other modified nucleobases suchas 8-substituted purines, xanthine, or hypoxanthine (the latter twobeing the natural degradation products). The modified nucleobasesdisclosed in Chiu and Rana, R N A, 2003, 9, 1034-1048, Limbach et al.Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao,Comprehensive Natural Products Chemistry, vol. 7, 313, are alsocontemplated, the contents of which are incorporated herein byreference.

Further examples of base pairing moieties include, but are not limitedto, expanded-size nucleobases in which one or more benzene rings hasbeen added. Nucleic base replacements described in the Glen Researchcatalog (www.glenresearch.com); Krueger A T et al., Acc. Chem. Res.,2007, 40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; BennerS. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., etal., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin.Chem. Biol., 2006, 10, 622-627, the contents of which are incorporatedherein by reference, are contemplated as useful for the synthesis of theoligomers described herein. Examples of expanded-size nucleobases areshown below:

The terms “oligonucleotide” or “oligomer” refer to a compound comprisinga plurality of linked nucleosides, nucleotides, or a combination of bothnucleosides and nucleotides. In specific embodiments provided herein, anoligonucleotide is a morpholino oligonucleotide.

The phrase “morpholino oligonucleotide” or “PMO” refers to a modifiedoligonucleotide having morpholino subunits linked together byphosphoramidate or phosphorodiamidate linkages, joining the morpholinonitrogen of one subunit to the 5′-exocyclic carbon of an adjacentsubunit. Each morpholino subunit comprises a nucleobase-pairing moietyeffective to bind, by nucleobase-specific hydrogen bonding, to anucleobase in a target.

The terms “antisense oligomer,” “antisense compound” and “antisenseoligonucleotide” are used interchangeably and refer to a sequence ofsubunits, each bearing a base-pairing moiety, linked by intersubunitlinkages that allow the base-pairing moieties to hybridize to a targetsequence in a nucleic acid (typically an RNA) by Watson-Crick basepairing, to form a nucleic acid:oligomer heteroduplex within the targetsequence. The oligomer may have exact (perfect) or near (sufficient)sequence complementarity to the target sequence; variations in sequencenear the termini of an oligomer are generally preferable to variationsin the interior.

Such an antisense oligomer can be designed to block or inhibittranslation of mRNA or to inhibit/alter natural or abnormal pre-mRNAsplice processing, and may be said to be “directed to” or “targetedagainst” a target sequence with which it hybridizes. The target sequenceis typically a region including an AUG start codon of an mRNA, aTranslation Suppressing Oligomer, or splice site of a pre-processedmRNA, a Splice Suppressing Oligomer (SSO). The target sequence for asplice site may include an mRNA sequence having its 5′ end 1 to about 25base pairs downstream of a normal splice acceptor junction in apreprocessed mRNA. In various embodiments, a target sequence may be anyregion of a preprocessed mRNA that includes a splice site or iscontained entirely within an exon coding sequence or spans a spliceacceptor or donor site. An oligomer is more generally said to be“targeted against” a biologically relevant target, such as a protein,virus, or bacteria, when it is targeted against the nucleic acid of thetarget in the manner described above.

The antisense oligonucleotide and the target RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother, such that stable and specific binding occurs between theoligonucleotide and the target. Thus, “specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof complementarity or precise pairing such that stable and specificbinding occurs between the oligonucleotide and the target. It isunderstood in the art that the sequence of an oligonucleotide need notbe 100% complementary to that of its target sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target molecule interferes withthe normal function of the target RNA, and there is a sufficient degreeof complementarity to avoid non-specific binding of the antisenseoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, and in the case of invitro assays, under conditions in which the assays are performed.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. Oligonucleotidescontaining a modified or substituted base include oligonucleotides inwhich one or more purine or pyrimidine bases most commonly found innucleic acids are replaced with less common or non-natural bases. Insome embodiments, the nucleobase is covalently linked at the N9 atom ofthe purine base, or at the N1 atom of the pyrimidine base, to themorpholine ring of a nucleotide or nucleoside.

Purine bases comprise a pyrimidine ring fused to an imidazole ring, asdescribed by the general formula:

Adenine and guanine are the two purine nucleobases most commonly foundin nucleic acids. These may be substituted with othernaturally-occurring purines, including but not limited toN6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

Pyrimidine bases comprise a six-membered pyrimidine ring as described bythe general formula:

Cytosine, uracil, and thymine are the pyrimidine bases most commonlyfound in nucleic acids. These may be substituted with othernaturally-occurring pyrimidines, including but not limited to5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and4-thiouracil. In one embodiment, the oligonucleotides described hereincontain thymine bases in place of uracil.

Other modified or substituted bases include, but are not limited to,2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine(e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives,5-substituted pyrimidine (e.g. 5-halouracil, 5-propynyluracil,5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil,5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T),7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine,8-aza-7-deazaguanine, 8-aza-7-deazaadenine,8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine,or derivatives thereof; N2-cyclopentylguanine (cPent-G),N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine(Pr-AP), pseudouracil or derivatives thereof; and degenerate oruniversal bases, like 2,6-difluorotoluene or absent bases like abasicsites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose;or pyrrolidine derivatives in which the ring oxygen has been replacedwith nitrogen (azaribose)). Pseudouracil is a naturally occurringisomerized version of uracil, with a C-glycoside rather than the regularN-glycoside as in uridine.

Certain modified or substituted nucleobases are particularly useful forincreasing the binding affinity of the antisense oligonucleotides of thedisclosure. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. Invarious embodiments, nucleobases may include 5-methylcytosinesubstitutions, which have been shown to increase nucleic acid duplexstability by 0.6-1.2° C.

In some embodiments, modified or substituted nucleobases are useful forfacilitating purification of antisense oligonucleotides. For example, incertain embodiments, antisense oligonucleotides may contain three ormore (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In certainantisense oligonucleotides, a string of three or more consecutiveguanine bases can result in aggregation of the oligonucleotides,complicating purification. In such antisense oligonucleotides, one ormore of the consecutive guanines can be substituted with hypoxanthine.The substitution of hypoxanthine for one or more guanines in a string ofthree or more consecutive guanine bases can reduce aggregation of theantisense oligonucleotide, thereby facilitating purification.

The oligonucleotides provided herein are synthesised and do not includeantisense compositions of biological origin. The molecules of thedisclosure may also be mixed, encapsulated, conjugated or otherwiseassociated with other molecules, molecule structures or mixtures ofcompounds, as for example, liposomes, receptor targeted molecules, oral,rectal, topical or other formulations, for assisting in uptake,distribution, or absorption, or a combination thereof.

The terms “complementary” and “complementarity” refer tooligonucleotides (i.e., a sequence of nucleotides) related bybase-pairing rules. For example, the sequence “T-G-A (5′-3′),” iscomplementary to the sequence “T-C-A (5′-3′).” Complementarity may be“partial,” in which only some of the nucleic acids' bases are matchedaccording to base pairing rules. Or, there may be “complete,” “total,”or “perfect” (100%) complementarity between the nucleic acids. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. While perfect complementarity is often desired, someembodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1mismatches with respect to the target RNA. Such hybridization may occurwith “near” or “substantial” complementarity of the antisense oligomerto the target sequence, as well as with exact complementarity. In someembodiments, an oligomer may hybridize to a target sequence at about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%complementarity. Variations at any location within the oligomer areincluded. In certain embodiments, variations in sequence near thetermini of an oligomer are generally preferable to variations in theinterior, and if present are typically within about 6, 5, 4, 3, 2, or 1nucleotides of the 5′-terminus, 3′-terminus, or both termini.

The term “peptide” refers to a compound comprising a plurality of linkedamino acids. The peptides provided herein can be considered to be cellpenetrating peptides.

The terms “cell penetrating peptide” and “CPP” are used interchangeablyand refer to cationic cell penetrating peptides, also called transportpeptides, carrier peptides, or peptide transduction domains. Thepeptides, provided herein, have the capability of inducing cellpenetration within 100% of cells of a given cell culture population andallow macromolecular translocation within multiple tissues in vivo uponsystemic administration. In various embodiments, a CPP embodiment of thedisclosure may include an arginine-rich peptide as described furtherbelow.

The term “treatment” refers to the application of one or more specificprocedures used for the amelioration of a disease. In certainembodiments, the specific procedure is the administration of one or morepharmaceutical agents. “Treatment” of an individual (e.g. a mammal, suchas a human) or a cell is any type of intervention used in an attempt toalter the natural course of the individual or cell. Treatment includes,but is not limited to, administration of a pharmaceutical composition,and may be performed either prophylactically or subsequent to theinitiation of a pathologic event or contact with an etiologic agent.Treatment includes any desirable effect on the symptoms or pathology ofa disease or condition, and may include, for example, minimal changes orimprovements in one or more measurable markers of the disease orcondition being treated. Also included are “prophylactic” treatments,which can be directed to reducing the rate of progression of the diseaseor condition being treated, delaying the onset of that disease orcondition, or reducing the severity of its onset. An “effective amount”or “therapeutically effective amount” refers to an amount of therapeuticcompound, such as an antisense oligomer, administered to a mammaliansubject, either as a single dose or as part of a series of doses, whichis effective to produce a desired therapeutic effect.

The term “amelioration” means a lessening of severity of at least oneindicator of a condition or disease. In certain embodiments,amelioration includes a delay or slowing in the progression of one ormore indicators of a condition or disease. The severity of indicatorsmay be determined by subjective or objective measures which are known tothose skilled in the art.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed oligonucleotides wherein the parentoligonucleotide is modified by converting an existing acid or basemoiety to its salt form. Lists of suitable salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977), each of which is incorporated herein by reference in itsentirety.

Peptide-Oligonucleotide-Conjugates

Provided herein are oligonucleotides chemically-linked to one or moremoieties, such as a cell penetrating peptide, that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Theoligonucleotides can additionally be chemically-linked to one or moreheteroalkyl moieties (e.g., polyethylene glycol) that further enhancethe activity, cellular distribution, or cellular uptake of theoligonucleotide. In one exemplary embodiment, the arginine-richpolypeptide is covalently coupled at its N-terminal or C-terminalresidue to either end, or both ends, of the antisense compound.

Thus, in one aspect, provided herein is apeptide-oligonucleotide-conjugate of Formula I:

or a pharmaceutically acceptable salt thereof,wherein:A′ is selected from —NHCH₂C(O)NH₂, —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

wherein

-   -   R⁵ is —C(O)(O-alkyl)_(x)-OH, wherein x is 3-10 and each alkyl        group is independently at each occurrence C₂₋₆-alkyl, or R⁵ is        selected from —C(O)C₁₋₆ alkyl, trityl, monomethoxytrityl,        —(C₁₋₆-alkyl)R⁶, —(C₁₋₆ heteroalkyl)-R⁶, aryl-R⁶, heteroaryl-R⁶,        —C(O)O—(C₁₋₆ alkyl)-R⁶, —C(O)O-aryl-R⁶, —C(O)O— heteroaryl-R⁶,        and

-   -   -   wherein R⁶ is selected from OH, SH, and NH₂, or R⁶ is O, S,            or NH, covalently linked to a solid support;

each R¹ is independently selected from OH and —NR³R⁴, wherein each R³and R⁴ are independently at each occurrence —C₁₋₆ alkyl;

each R² is independently selected from H, a nucleobase, and a nucleobasefunctionalized with a chemical protecting-group, wherein the nucleobaseindependently at each occurrence comprises a C₃₋₆ heterocyclic ringselected from pyridine, pyrimidine, triazinane, purine, anddeaza-purine;

z is 8-40; and

E′ is selected from H, —C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, benzoyl, stearoyl,trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

-   -   wherein        -   Q is —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,        -   R⁷ is —(CH₂)₂OC(O)N(R⁸)₂, wherein R⁸ is —(CH₂)₆NHC(═NH)NH₂,            and        -   R¹¹ is selected from OH and —NR³R⁴,

wherein L is covalently linked by an amide bond to the carboxy-terminusof J, and L is selected from —NH(CH₂)₁₋₆C(O)—,—NH(CH₂)₁₋₆C(O)NH(CH₂)₁₋₆C(O)—, and

t is 4-9;

each J is independently at each occurrence selected from an amino acidof the structure

-   -   wherein:        -   r and q are each independently 0, 1, 2, 3, or 4; and        -   each R⁹ is independently at each occurrence selected from H,            an amino acid side-chain, and an amino acid side-chain            functionalized with a chemical protecting-group,        -   wherein two or more amino acid side-chain groups of R⁹            independently at each occurrence comprise a sulfur, wherein            two of the sulfur atoms, together with the atoms to which            they are attached, form the structure

-   -   -   -   wherein d is 0 or 1, and M is selected from:

-   -   wherein each R¹⁰ is independently at each occurrence H or a        halogen; and    -   G is covalently linked to the amino-terminus of J, and G is        selected from    -   H, —C(O)C₁₋₆ alkyl, benzoyl, and stearoyl, and

wherein at least one of the following conditions is true:

-   -   A′ is

2) E′ is

or 3) E′ is

In one embodiment, E′ is selected from H, —C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl,benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl,trimethoxytrityl, and

In another embodiment, only one of A′ is

E′ is

or E′ is

In still another embodiment, A′ is

or E′ is

In yet another embodiment, A′ is selected from —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

In another embodiment E′ is selected from H, —C(O)CH₃, trityl,4-methoxytrityl, benzoyl, stearoyl, and

In still another embodiment, A′ is selected from—N(C₁₋₆-alkyl)CH₂C(O)NH₂,

In yet another embodiment, A′ is

In another embodiment, E′ is selected from H, —C(O)CH₃, trityl,4-methoxytrityl, benzoyl, and stearoyl.

In yet another embodiment, E′ is selected from H and —C(O)CH₃.

In still another embodiment, the peptide-oligonucleotide-conjugate ofFormula I is a peptide-oligonucleotide-conjugate of Formula Ia:

In an embodiment of Formula I and Ia, R⁵ is —C(O)(O-alkyl)_(x)OH,wherein each alkyl group is independently for each occurrenceC₂₋₆-alkyl.

In another embodiment of Formula I and Ia, R⁵ is —C(O)(O—CH₂CH₂)₃OH.

In another embodiment, the peptide-oligonucleotide-conjugate of FormulaI is a peptide-oligonucleotide-conjugate of Formula Ib:

In an embodiment of Formula I and Ib, E′ is selected from H, C₁₋₆ alkyl,—C(O)CH₃, benzoyl, and stearoyl.

In another embodiment of Formula I and Ib, E′ is selected from H and—C(O)CH₃.

In an embodiment of Formula I, Ia, and Ib, each R¹⁰ is independently ahalogen selected from fluorine, chlorine, bromine, and iodine.

In another embodiment of Formula I, Ia, and Ib, each R¹⁰ is fluorine.

In still another embodiment of Formula I, Ia, and Ib, M is

In another embodiment of Formula I, Ia, and Ib, two amino acidside-chain groups are independently at each occurrence cysteine orhomocysteine amino acid side-chain groups.

In still another embodiment of Formula I, Ia, and Ib, each J isindependently at each occurrence selected from an α-amino acid, aβ²-amino acid, and a β³-amino acid.

In yet another embodiment of Formula I, Ia, and Ib, r and q are each 0.

In another embodiment of Formula I, Ia, and Ib, J is independentlyselected from cysteine and arginine.

In still another embodiment of Formula I, Ia, and Ib, two J groups arecysteine.

In yet another embodiment of Formula I, Ia, and Ib, z is 8-25.

In another embodiment of Formula I, Ia, and Ib, z is 15-25.

In still another embodiment of Formula I, Ia, and Ib, z is 10-20.

In another embodiment of Formula I, Ia, and Ib, each Ie is independentlyNR³R⁴, wherein each R³ and R⁴ are independently at each occurrenceC₁₋₃-alkyl.

In still another embodiment of Formula I, Ia, and Ib, each R¹ isN(CH₃)₂.

In yet another embodiment of Formula I, Ia, and Ib, each R² is anucleobase, wherein the nucleobase independently at each occurrencecomprises a C₄₋₆-heterocyclic ring selected from pyridine, pyrimidine,triazinane, purine, and deaza-purine.

In another embodiment of Formula I, Ia, and Ib, each R² is a nucleobase,wherein the nucleobase independently at each occurrence comprises aC₄₋₆-heterocyclic ring selected from pyrimidine, purine, anddeaza-purine.

In still another embodiment of Formula I, Ia, and Ib, each R² is anucleobase independently at each occurrence selected from adenine,2,6-diaminopurine, 7-deaza-adenine, guanine, 7-deaza-guanine,hypoxanthine, cytosine, 5-methyl-cytosine, thymine, uracil, andhypoxanthine.

In yet another embodiment of Formula I, Ia, and Ib, each R² is anucleobase independently at each occurrence selected from adenine,guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.

In another embodiment of Formula I, Ia, and Ib, L is selected from—NH(CH₂)₁₋₆C(O)—, —NH(CH₂)₅C(O)NH(CH₂)₂C(O)—, and

In still another embodiment of Formula I, Ia, and Ib, L is selected fromglycine and

In yet another embodiment of Formula I, Ia, and Ib, L is glycine.

In another embodiment of Formula I, Ia, and Ib, G is selected from H,C(O)CH₃, benzoyl, and stearoyl.

In still another embodiment of Formula I, Ia, and Ib, G is H or—C(O)CH₃.

In yet another embodiment of Formula I, Ia, and Ib, G is —C(O)CH₃.

In another embodiment of Formula I, Ia, and Ib, d is 1.

In still another embodiment of Formula I, Ia, and Ib, d is 0.

In yet another embodiment of Formula I, Ia, and Ib,

is selected from:

In still another embodiment, the peptide-oligonucleotide-conjugate ofFormula I is a peptide-oligonucleotide-conjugate of Formula Ic:

In yet another embodiment, the peptide-oligonucleotide-conjugate ofFormula I is a peptide-oligonucleotide-conjugate of Formula Id:

In another embodiment, the peptide-oligonucleotide-conjugate of FormulaI is a peptide-oligonucleotide-conjugate of Formula Ie:

In another aspect, provided herein is apeptide-oligonucleotide-conjugate of Formula IV:

or a pharmaceutically acceptable salt thereof,wherein:A′ is selected from —NHCH₂C(O)NH₂, —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

wherein

-   -   R⁵ is —C(O)(O-alkyl)_(x)-OH, wherein x is 3-10 and each alkyl        group is independently at each occurrence C₂₋₆-alkyl, or R⁵ is        selected from —C(O)C₁₋₆ alkyl, trityl, monomethoxytrityl,        —(C₁₋₆-alkyl)R⁶, —(C₁₋₆ heteroalkyl)-R⁶, aryl-R⁶, heteroaryl-R⁶,        —C(O)O—(C₁₋₆ alkyl)-R⁶, —C(O)O-aryl-R⁶, —C(O)O— heteroaryl-R⁶,        and

-   -   -   wherein R⁶ is selected from OH, SH, and NH₂, or R⁶ is O, S,            or NH,        -   covalently linked to a solid support;

each R¹ is independently selected from OH and —NR³R⁴, wherein each R³and R⁴ are independently at each occurrence —C₁₋₆ alkyl;

each R² is independently selected from H, a nucleobase, and a nucleobasefunctionalized with a chemical protecting-group, wherein the nucleobaseindependently at each occurrence comprises a C₃₋₆ heterocyclic ringselected from pyridine, pyrimidine, triazinane, purine, anddeaza-purine;

z is 8-40; and

E′ is selected from H, —C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, benzoyl, stearoyl,trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

-   -   wherein        -   Q is —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,        -   R⁷ is —(CH₂)₂OC(O)N(R⁸)₂, wherein R⁸ is —(CH₂)₆NHC(═NH)NH₂,            and        -   R¹¹ is selected from OH and —NR³R⁴,

wherein L is covalently linked by an amide bond to the carboxy-terminusof J, and L is selected from —NH(CH₂)₁₋₆C(O)—,—NH(CH₂)₁₋₆C(O)NH(CH₂)₁₋₆C(O)—, and

t is 4-9;

each J is independently at each occurrence selected from an amino acidof the structure

-   -   wherein:        -   r and q are each independently 0, 1, 2, 3, or 4; and        -   each R⁹ is independently at each occurrence selected from H,            an amino acid side-chain, and an amino acid side-chain            functionalized with a chemical protecting-group,        -   wherein two or more amino acid side-chain groups of R⁹            independently at each occurrence comprise a sulfur, wherein            two of the sulfur atoms, together with the atoms to which            they are attached, form the structure

and

-   -   G is covalently linked to the amino-terminus of J, and G is        selected from H, —C(O)C₁₋₆ alkyl, benzoyl, and stearoyl, and

wherein at least one of the following conditions is true:

-   -   1) A′ is

2) E′ is

or 3) E′ is

In an embodiment of Formula IV, d is 1.

In another embodiment of Formula IV, E′ is selected from H, —C(O)CH₃,and

In still another embodiment of Formula IV, A′ is selected from—N(C₁₋₆-alkyl)CH₂C(O)NH₂,

In yet another embodiment of Formula IV, A′ is

In another embodiment of Formula IV, E′ is selected from H and —C(O)CH₃.

In still another embodiment of Formula IV, M is

In still another embodiment of Formula IV, each J is independently ateach occurrence selected from an α-amino acid, a β²-amino acid, and aβ³-amino acid.

In yet another embodiment of Formula IV, r and q are each 0.

In another embodiment of Formula IV, J is independently selected fromcysteine and arginine.

In another embodiment of Formula IV, z is 15-25.

In yet another embodiment of Formula IV, z is 10-20.

In still another embodiment of Formula IV, each R¹ is N(CH₃)₂.

In yet another embodiment of Formula IV, each R² is a nucleobaseindependently at each occurrence selected from adenine, guanine,cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.

In yet another embodiment of Formula IV, L is glycine.

In another embodiment of Formula IV, G is selected from H, C(O)CH₃,benzoyl, and stearoyl.

In yet another embodiment of Formula IV, G is —C(O)CH₃.

In yet another embodiment, the oligonucleotide comprises a targetingsequence having sequence complementarity to an RNA target. In a specificembodiment, the RNA target is a cellular RNA target. In another specificembodiment, the targeting sequence has sufficient sequencecomplementarity to bind to the RNA target. In yet another specificembodiment, the targeting sequence has perfect sequence complementarityto the RNA target.

Representative

moieties of the disclosure include, amongst others, moieties of thefollowing formulae:

Representative peptide-oligonucleotide-conjugates of the disclosureinclude, amongst others, peptide-oligonucleotide-conjugates of thefollowing structures:

or a pharmaceutically acceptable salt thereof, wherein

G is selected from H and —C(O)CH₃, and

E′ is selected from H and —C(O)CH₃.

In one embodiment of the peptide-oligonucleotide-conjugates of thedisclosure, G is H.

In another embodiment of the peptide-oligonucleotide-conjugates of thedisclosure, G is —C(O)CH₃.

In still another embodiment of the peptide-oligonucleotide-conjugates ofthe disclosure, E′ is H.

In yet another embodiment of the peptide-oligonucleotide-conjugates ofthe disclosure, E′ is —C(O)CH₃.

In still another embodiment of the peptide-oligonucleotide-conjugates ofthe disclosure, E′ and G are —C(O)CH₃.

In yet another embodiment of the peptide-oligonucleotide-conjugates ofthe disclosure, G is —C(O)CH₃ and E′ is H.

In some embodiments, the peptide-oligonucleotide-conjugates describedherein are unsolvated. In other embodiments, one or more of thepeptide-oligonucleotide-conjugates are in solvated form. As known in theart, the solvate can be any of pharmaceutically acceptable solvent, suchas water, ethanol, and the like.

Although the peptide-oligonucleotide-conjugates of Formulae I, Ia, Ib,Ic, Id, Ie, and IV are depicted in their neutral forms, in someembodiments, these peptide-oligonucleotide-conjugates are used in apharmaceutically acceptable salt form.

Oligonucleotides

Important properties of morpholino-based subunits include: 1) theability to be linked in a oligomeric form by stable, uncharged orpositively charged backbone linkages; 2) the ability to support anucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil,5-methyl-cytosine and hypoxanthine) such that the polymer formed canhybridize with a complementary-base target nucleic acid, includingtarget RNA, T_(M) values above about 45° C. in relatively shortoligonucleotides (e.g., 10-15 bases); 3) the ability of theoligonucleotide to be actively or passively transported into mammaliancells; and 4) the ability of the oligonucleotide and oligonucleotide:RNAheteroduplex to resist RNAse and RNase H degradation, respectively.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding T_(M) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(M) of an oligomer withrespect to complementary-sequence RNA may be measured by conventionalmethods, such as those described by Hames et al., Nucleic AcidHybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C.G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, MethodsEnzymol. Vol. 154 pp. 94-107. In certain embodiments, antisenseoligomers may have a binding T_(M), with respect to acomplementary-sequence RNA, of greater than body temperature and, insome embodiments greater than about 45° C. or 50° C. T_(M)'s in therange 60-80° C. or greater are also included. According to well-knownprinciples, the T_(M) of an oligomer, with respect to acomplementary-based RNA hybrid, can be increased by increasing the ratioof C:G paired bases in the duplex, or by increasing the length (in basepairs) of the heteroduplex, or both. At the same time, for purposes ofoptimizing cellular uptake, it may be advantageous to limit the size ofthe oligomer. For this reason, compounds of the disclosure includecompounds that show a high T_(M) (45-50° C. or greater) at a length of25 bases or less.

The length of an oligonucleotide may vary so long as it is capable ofbinding selectively to the intended location within the pre-mRNAmolecule. The length of such sequences can be determined in accordancewith selection procedures described herein. Generally, theoligonucleotide will be from about 8 nucleotides in length up to about50 nucleotides in length. For example, the length of the oligonucleotide(z) can be 8-38, 8-25, 15-25, 17-21, or about 18. It will be appreciatedhowever that any length of nucleotides within this range may be used inthe methods described herein.

In some embodiments, the antisense oligonucleotides contain basemodifications or substitutions. For example, certain nucleo-bases may beselected to increase the binding affinity of the antisenseoligonucleotides described herein. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine and2,6-diaminopurine. 5-methylcytosine substitutions have been shown toincrease nucleic acid duplex stability by 0.6-1.2° C., and may beincorporated into the antisense oligonucleotides described herein. Inone embodiment, at least one pyrimidine base of the oligonucleotidecomprises a 5-substituted pyrimidine base, wherein the pyrimidine baseis selected from the group consisting of cytosine, thymine and uracil.In one embodiment, the 5-substituted pyrimidine base is5-methylcytosine. In another embodiment, at least one purine base of theoligonucleotide comprises an N-2, N-6 substituted purine base. In oneembodiment, the N-2, N-6 substituted purine base is 2, 6-diaminopurine.

Morpholino-based oligomers (including antisense oligomers) are detailed,for example, in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047;5,034,506; 5,166,315; 5,185,444; 5,521,063; 5,506,337 and pending U.S.patent application Ser. Nos. 12/271,036; 12/271,040; and PCT PublicationNo. WO/2009/064471 and WO/2012/043730 and Summerton et al. 1997,Antisense and Nucleic Acid Drug Development, 7, 187-195, which arehereby incorporated by reference in their entirety.

Accordingly, in one aspect, provided herein is an oligonucleotide ofFormula II:

or a pharmaceutically acceptable salt thereof,

wherein

A is selected from the group consisting of OH, —NHCH₂C(O)NH₂,—N(C₁₋₆-alkyl)CH₂C(O)NH₂, and

R⁵ is —C(O)(O-alkyl)_(x)OH, wherein x is 3-10 and each alkyl group isindependently at each occurrence —C₂₋₆-alkyl, or R⁵ is selected from thegroup consisting of —C(O)C₁₋₆-alkyl, trityl, monomethoxytrityl,—C₁₋₆-alkyl-R⁶, —C₁₋₆-heteroalkyl-R⁶, -aryl-R⁶, -heteroaryl-R⁶,—C(O)O—C₁₋₆-alkyl-R⁶, —C(O)O-aryl-R⁶, and —C(O)O-heteroaryl-R⁶;

R⁶ is selected from the group consisting of OH, SH, and NH₂, or R⁶ is O,S, or NH, covalently linked to a solid support;

each R¹ is independently OH or —NR³R⁴;

each R³ and R⁴ are independently at each occurrence —C₁₋₆-alkyl;

each R² is independently selected from the group consisting of H, anucleobase, and a nucleobase functionalized with a chemicalprotecting-group, wherein the nucleobase independently at eachoccurrence comprises a C₃₋₆-heterocyclic ring selected from the groupconsisting of pyridine, pyrimidine, triazinane, purine, anddeaza-purine;

z is 8-40;

E is selected from the group consisting of H, —C₁₋₆-alkyl,—C(O)C₁₋₆-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl,

Q is —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—;

R⁷ is —(CH₂)₂OC(O)N(R⁸)₂;

R⁸ is —(CH₂)₆NHC(═NH)NH₂;

R¹² is —C(O)NHC₁₋₆-alkyl or —C(O)OC₁₋₆-alkyl; and

R¹³ is selected from the group consisting of trityl, monomethoxytrityl,dimethoxytrityl, and trimethoxytrityl.

In one embodiment of Formula II, A is

E is selected from the group consisting of H, —C(O)CH₃, benzoyl, andstearoyl;

R⁵ is —C(O)(O-alkyl)_(x)-OH, wherein each alkyl group is independentlyat each occurrence —C₂₋₆-alkyl, trityl, and 4-methoxytrityl; and

each R² is independently a nucleobase, wherein the nucleobaseindependently at each occurrence comprises a C₄₋₆-heterocyclic ringselected from the group consisting of pyridine, pyrimidine, purine, anddeaza-purine.

In another embodiment of Formula II, R⁵ is C(O)(O—CH₂CH₂)₃—OH; and

each R² is independently a nucleobase, wherein the nucleobaseindependently at each occurrence comprises a pyrimidine or a purine.

In still another embodiment, the oligonucleotide of Formula II is anoligonucleotide of Formula IIa:

In an embodiment of Formula II and IIa, R² is independently at eachoccurrence adenine, 2,6-diaminopurine, guanine, hypoxanthine, cytosine,5-methyl-cytosine, thymine, uracil, and hypoxanthine; and

each R¹ is —N(CH₃)₂.

Provided in Table 1 are various embodiments of nucleotide moieties asdescribed herein.

TABLE 1 Various embodiments of nucleotide moieties.

In some embodiments, the oligonucleotides described herein areunsolvated. In other embodiments, one or more of the oligonucleotidesare in solvated form. As known in the art, the solvate can be any ofpharmaceutically acceptable solvent, such as water, ethanol, and thelike.

Although the oligonucleotides of Formulas II and IIa, are depicted intheir neutral forms, in some embodiments, these oligonucleotides areused in a pharmaceutically acceptable salt form.

Peptides

The oligonucleotides provided herein include an oligonucleotide moietyconjugated to a CPP. In some embodiments, the CPP can be anarginine-rich peptide transport moiety effective to enhance transport ofthe compound into cells. The transport moiety is, in some embodiments,attached to a terminus of the oligomer. The peptides have the capabilityof inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or100% of cells of a given cell culture population, including all integersin between, and allow macromolecular translocation within multipletissues in vivo upon systemic administration. In one embodiment, thecell-penetrating peptide may be an arginine-rich peptide transporter. Invarious embodiments, a peptide-oligonucleotide-conjugate of the presentdisclosure may utilize glycine as the linker between the CPP and theantisense oligonucleotide.

The transport moieties as described above have been shown to greatlyenhance cell entry of attached oligomers, relative to uptake of theoligomer in the absence of the attached transport moiety. Uptake may beenhanced at least ten fold, and, in some embodiments, twenty fold,relative to the unconjugated compound.

The use of arginine-rich peptide transporters (i.e., cell-penetratingpeptides) are particularly useful in practicing the present disclosure.Certain peptide transporters have been shown to be highly effective atdelivery of antisense compounds into primary cells including musclecells. Furthermore, compared to other known peptide transporters such asPenetratin and the Tat peptide, the peptide transporters describedherein, when conjugated to an antisense PMO, demonstrate an enhancedability to alter splicing of several gene transcripts.

Thus, in one aspect, provided herein is a peptide of Formula III:

G-(J)_(t)-L   (III)

or a pharmaceutically acceptable salt thereof,

wherein

G is selected from the group consisting of H, C(O)C₁₋₆-alkyl, benzoyl,and stearoyl;

each J is independently at each occurrence selected from an amino acidof the structure

each R⁹ is independently at each occurrence selected from the groupconsisting of H, an amino acid side-chain, and an amino acid side-chainfunctionalized with a chemical protecting-group;

wherein two or more amino acid side-chain groups of R⁹ independently ateach occurrence comprise a thiol or a thiol functionalized with achemical protecting-group;

r and q are independently 0, 1, 2, 3, or 4;

L is selected from the group consisting of —NH(CH₂)₁₋₆C(O)OH,—NH(CH₂)₅C(O)NH(CH₂)₂C(O)OH, and

each of which may be covalently-linked to a solid support; and

t is 4-9.

In one embodiment, two amino acid side-chain groups, wherein each of thetwo amino acid side-chain groups independently comprise a sulfur,together with the atoms to which they are attached, form the structure

M is selected from the group consisting of

and

d is 0 or 1.

In another embodiment, M is

In yet another embodiment, two amino acid side-chain groups areindependently at each occurrence cysteine or homocysteine amino acidside-chain groups.

In still another embodiment, each J is independently at each occurrenceselected from an α-amino acid, a β²-amino acid, and a β³-amino acid.

In another embodiment, r and q are each 0.

In another embodiment, J is independently selected from cysteine andarginine.

In yet another embodiment, two J groups are cysteine.

In still another embodiment, L is selected from —NH(CH₂)₁₋₆C(O)OH and

In another embodiment, L is

In yet another embodiment, L is NHCH₂C(O)OH.

In yet another embodiment, G is selected from the group consisting of H,C(O)CH₃, benzoyl, and stearoyl.

In still another embodiment, G is C(O)CH₃ or stearoyl.

In another embodiment, G is C(O)CH₃.

In yet another embodiment, G is covalently linked to the amino-terminusof J. In a further embodiment, L is covalently linked by an amide bondto the carboxy-terminus of J.

In another embodiment, d is 0.

In yet another embodiment, d is 1.

Provided in Table 2 are representative peptides as described herein.

TABLE 2 Exemplary Peptides Structure Compound SEQ ID NO

1AC 1AD 1AE 1 1BC   1BD   1BE  

2AC 2AD 2AE 2 2BC   2BD   2BE  

3AC 3AD 3AE 3 3BC   3BD   3BE  

4AC 4AD 4AE 4 4BC   4BD   4BE  

5AC 5AD 5AE 5 5BC   5BD   5BE  

6AC 6AD 6AE 6 6BC   6BD   6BE  

7AC 7AD 7AE 7 7BC   7BD   7BE  

8AC 8AD 8AE 8 8BC   8BD   8BE   Gly = glycinyl or Gly-P3P    (A)   (B) R= Arg  

In some embodiments, the peptides described herein are unsolvated. Inother embodiments, one or more of the peptides are in solvated form. Asknown in the art, the solvate can be any of pharmaceutically acceptablesolvent, such as water, ethanol, and the like.

Although the peptides of Formula III, are depicted in their neutralforms, in some embodiments, these oligonucleotides are used in apharmaceutically acceptable salt form.

General Synthetic Schemes

The process of Scheme IIa is applicable to conjugation of the peptide tothe 5′ end of the PMO under similar conditions (see Scheme Ic).

The process of Scheme IIb is applicable to conjugation of the peptide tothe 5′ end of the PMO under similar conditions.

Methods

Provided herein are methods of treating a muscle disease, a viralinfection, or a bacterial infection in a subject in need thereof,comprising administering to the subject apeptide-oligonucleotide-conjugate of Formulae I, Ia, Ib, Ic, Id, Ie, orV.

Accordingly, in one aspect, provided herein is a method of treating amuscle disease, a viral infection, or a bacterial infection in a subjectin need thereof, comprising administering to the subject apeptide-oligonucleotide-conjugate of the present disclosure.

In one embodiment, the muscle disease is Duchenne Muscular Dystrophy.

In another embodiment, the viral infection is caused by a virus selectedfrom the group consisting of marburg virus, ebola virus, influenzavirus, and dengue virus.

In another embodiment, the bacterial infection is caused byMycobacterium tuberculosis.

The subject considered herein is typically a human. However, the subjectcan be any mammal for which treatment is desired. Thus, the methodsdescribed herein can be applied to both human and veterinaryapplications.

Administration/Dose

The formulation of therapeutic compositions and their subsequentadministration (dosing) is within the skill of those in the art. Dosingis dependent on severity and responsiveness of the disease state to betreated, with the course of treatment lasting from several days toseveral months, or until a sufficient diminution of the disease state isachieved. Optimal dosing schedules can be calculated from measurementsof drug accumulation in the body of the patient.

Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligomers, and can generally beestimated based on EC₅₀s found to be effective in in vitro and in vivoanimal models. In general, dosage is from 0.01 μg to 100 g/kg of bodyweight, and may be given once or more daily, weekly, monthly or yearly,or even once every 2 to 20 years. Persons of ordinary skill in the artcan easily estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thepatient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligomer is administered in maintenancedoses, ranging from 0.01 μg to 100 g/kg of body weight, once or moredaily, to once every 20 years.

In some embodiments, the oligonucleotide (an oligonucleotide of FormulaeII or IIa) is administered alone.

In some embodiments, the oligonucleotide is administered in atherapeutically effective amount or dosage. A “therapeutically effectiveamount” is an amount of an oligonucleotide of Formula II or IIa that,when administered to a patient by itself, effectively treats a muscledisease, a viral infection, or a bacterial infection. An amount thatproves to be a “therapeutically effective amount” in a given instance,for a particular subject, may not be effective for 100% of subjectssimilarly treated for the disease or condition under consideration, eventhough such dosage is deemed a “therapeutically effective amount” byskilled practitioners. The amount of the oligonucleotide thatcorresponds to a therapeutically effective amount is strongly dependenton the type of disease, stage of the disease, the age of the patientbeing treated, and other facts.

In different embodiments, depending on the oligonucleotide of FormulaeII or IIa and the effective amounts used, the oligonucleotides canmodulate the expression of a gene involved in a muscle disease, a viralinfection, or a bacterial infection.

While the amounts of an oligonucleotide of Formulae II or IIa shouldresult in the effective treatment of a muscle disease, a viralinfection, or a bacterial infection, the amounts, are preferably notexcessively toxic to the patient (i.e., the amounts are preferablywithin toxicity limits as established by medical guidelines). In someembodiments, either to prevent excessive toxicity or provide a moreefficacious treatment, or both, of a muscle disease, a viral infection,or a bacterial infection, a limitation on the total administered dosageis provided. Typically, the amounts considered herein are per day;however, half-day and two-day or three-day cycles also are consideredherein.

Different dosage regimens may be used to treat a muscle disease, a viralinfection, or a bacterial infection. In some embodiments, a dailydosage, such as any of the exemplary dosages described above, isadministered once, twice, three times, or four times a day for three,four, five, six, seven, eight, nine, or ten days. Depending on the stageand severity of the disease being treated, a shorter treatment time(e.g., up to five days) may be employed along with a high dosage, or alonger treatment time (e.g., ten or more days, or weeks, or a month, orlonger) may be employed along with a low dosage. In some embodiments, aonce- or twice-daily dosage is administered every other day.

Oligonucleotides of Formula II and IIa, or their pharmaceuticallyacceptable salts or solvate forms, in pure form or in an appropriatepharmaceutical composition, can be administered via any of the acceptedmodes of administration or agents known in the art. The oligonucleotidescan be administered, for example, orally, nasally, parenterally(intravenous, intramuscular, or subcutaneous), topically, transdermally,intravaginally, intravesically, intracistemally, or rectally. The dosageform can be, for example, a solid, semi-solid, lyophilized powder, orliquid dosage forms, such as for example, tablets, pills, soft elasticor hard gelatin capsules, powders, solutions, suspensions,suppositories, aerosols, or the like, for example, in unit dosage formssuitable for simple administration of precise dosages. A particularroute of administration is oral, particularly one in which a convenientdaily dosage regimen can be adjusted according to the degree of severityof the disease to be treated.

Auxiliary and adjuvant agents may include, for example, preserving,wetting, suspending, sweetening, flavoring, perfuming, emulsifying, anddispensing agents. Prevention of the action of microorganisms isgenerally provided by various antibacterial and antifungal agents, suchas, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonicagents, such as sugars, sodium chloride, and the like, may also beincluded. Prolonged absorption of an injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. The auxiliary agents also can includewetting agents, emulsifying agents, pH buffering agents, andantioxidants, such as, for example, citric acid, sorbitan monolaurate,triethanolamine oleate, butylated hydroxytoluene, and the like.

Solid dosage forms can be prepared with coatings and shells, such asenteric coatings and others well-known in the art. They can containpacifying agents and can be of such composition that they release theactive oligonucleotide or oligonucleotides in a certain part of theintestinal tract in a delayed manner. Examples of embedded compositionsthat can be used are polymeric substances and waxes. The activeoligonucleotides also can be in microencapsulated form, if appropriate,with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Suchdosage forms are prepared, for example, by dissolving, dispersing, etc.,the HDAC inhibitors or retinoic acid described herein, or apharmaceutically acceptable salt thereof, and optional pharmaceuticaladjuvants in a carrier, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol and the like; solubilizing agents andemulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propyleneglycol, 1,3-butyleneglycol, dimethyl formamide; oils, inparticular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols and fatty acid esters of sorbitan; or mixtures ofthese substances, and the like, to thereby form a solution orsuspension.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable compositions will contain about 1% to about99% by weight of the oligonucleotides described herein, or apharmaceutically acceptable salt thereof, and 99% to 1% by weight of apharmaceutically acceptable excipient. In one example, the compositionwill be between about 5% and about 75% by weight of a oligonucleotidedescribed herein, or a pharmaceutically acceptable salt thereof, withthe rest being suitable pharmaceutical excipients.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art. Reference is made, for example,to Remington's Pharmaceutical Sciences, 18th Ed. (Mack PublishingCompany, Easton, Pa., 1990).

Kits

In other embodiments, kits are provided. Kits according to thedisclosure include package(s) comprising oligonucleotides, peptides,peptide-oligonucleotide-conjugates, or compositions of the disclosure.In some embodiments, kits comprise a peptide-oligonucleotide-conjugateaccording to Formulae I, Ia, Ib, Ic, Id, Ie, or V, or a pharmaceuticallyacceptable salt thereof. In other embodiments, kits comprise anoligonucleotide according to Formulae II or IIa, or a pharmaceuticallyacceptable salt thereof. In still other embodiments, kits comprise apeptide according to Formula III, or a pharmaceutically acceptable saltthereof.

The phrase “package” means any vessel containing oligonucleotides orcompositions presented herein. In some embodiments, the package can be abox or wrapping. Packaging materials for use in packaging pharmaceuticalproducts are well-known to those of skill in the art. Examples ofpharmaceutical packaging materials include, but are not limited to,bottles, tubes, inhalers, pumps, bags, vials, containers, syringes,bottles, and any packaging material suitable for a selected formulationand intended mode of administration and treatment.

The kit can also contain items that are not contained within thepackage, but are attached to the outside of the package, for example,pipettes.

Kits can further contain instructions for administering oligonucleotidesor compositions of the disclosure to a patient. Kits also can compriseinstructions for approved uses of oligonucleotides herein by regulatoryagencies, such as the United States Food and Drug Administration. Kitscan also contain labeling or product inserts for the oligonucleotides.The package(s) or any product insert(s), or both, may themselves beapproved by regulatory agencies. The kits can include oligonucleotidesin the solid phase or in a liquid phase (such as buffers provided) in apackage. The kits can also include buffers for preparing solutions forconducting the methods, and pipettes for transferring liquids from onecontainer to another.

Examples

Examples have been set forth below for the purpose of illustration andto describe certain specific embodiments of the disclosure. However, thescope of the claims is not to be in any way limited by the examples setforth herein. Various changes and modifications to the disclosedembodiments will be apparent to those skilled in the art and suchchanges and modifications including, without limitation, those relatingto the chemical structures, substituents, derivatives, formulations ormethods of the disclosure may be made without departing from the spiritof the disclosure and the scope of the appended claims. Definitions ofthe variables in the structures in the schemes herein are commensuratewith those of corresponding positions in the formulae presented herein.

Example 1: A Protocol for Peptide Stapling with Hexafluorobenzene

To the cyclic disulfide peptide Ac-CRRRCRRRG-OH (74.8 mg, 38.5 μmop (SEQID NO:3) in a glass vial is added a solution of TRIS base (8 mL, 50 mMin DMF) and hexafluorobenzene (111 μL, 962 μmop. To this solution, TCEPsolution in H2O (136 μL, 0.71 M, pH=8) is added to reduce the disulfidebond and initiate reaction. The reaction mixture is stirred at roomtemperature for 7 hours and monitored by LC-MS. The resulting mixture isdiluted with 40 mL of 0.1% TFA in water, centrifuged, filtered, andsubjected to purification by HPLC. Fractions containing peptide product(analyzed by LC-MS) are combined and lyophilized to give the desiredperfluoroaryl peptide (19.9 mg, 8.9 μmol, 23% yield).

Example 2: A Protocol for Peptide Stapling with Decafluorobiphenyl

In a glass vial, the cyclic disulfide peptide Ac-CRRRCRRRG-OH (74.5 mg,38.4 mop (SEQ ID NO:3) and decafluorobiphenyl (25.6 mg, 76.7 mol aredissolved in a solution of TRIS base in DMF (8 mL, 50 mM). To thissolution is added a TCEP solution in H₂O (135 μL, 0.71 M, pH˜8) in orderto reduce the disulfide bond and initiate reaction. The reaction mixtureis stirred at room temperature for 7 hours and monitored by LC-MS. Theresulting mixture is diluted with TFA in water (40 mL of 0.1%),centrifuged, filtered, and subjected to purification by HPLC. Fractionscontaining peptide product are analyzed by LC-MS, combined, andlyophilized to give the final perfluoroaryl peptide (20 mg, 8.9 mol, 23%yield).

Example 3:

A Protocol for Peptide Conjugation

To a mixture of the PMO (nucleobase sequence (R²) is: GCT ATT ACC TTAACC CAG (SEQ ID NO:9); 10.7 mg, 1.72 μmop and the decafluorobiphenylpeptide Ac-CRRRCRRRG-OH (5.9 mg, 2.64 umol) in a plastic Eppendorf tubeis added DMSO (0.4 mL), HATU in DMSO (6.6 μL, 2.64 μmol, 0.4 M), andDIPEA (2.3 μL, 13.2 μmop. The contents of the tube are mixed and allowedto stand at room temperature for 2 hours. The resulting mixture isdiluted with water (8 mL) and subjected sequentially to weak cationexchange (CM Sepharose) and solid phase extraction (Amberchrom CG 300M).The resulting product is analyzed by LC-MS and MALDI-TOF MS, andlyophilized to give the PMO-peptide conjugate (9.8 mg, 1.26 μmol, 73%yield).

Example 4: A Protocol for Single Pot Conjugation of PMO with a CyclicDisulfide Peptide Followed by Peptide Stapling

To a solution of PMO (85 mg, 0.01 mmol, 1 eq; nucleobase sequence (R²):GCT ATT ACC TTA ACC CAG (SEQ ID NO:9)), the peptide(Ac-Cys-Arg-Arg-Arg-Arg-Arg-Arg-Cys-Gly-OH orAc-Cys-Arg-Arg-Arg-Cys-Arg-Arg-Arg-Gly-OH, 37 mg, each with a disulfidebridge, 2 eq) and HATU (8 mg, 2 eq) in DMSO (2 mL) is addeddiisopropylethylamine (9 μL, 5 eq). The reaction is stirred at roomtemperature for 5 hours. After adding 50 mM of dithiothreitol (DTT) inDMSO solution (1 mL, 5 eq), 50 mM of TRIS in DMSO solution (1 mL, 5 eq),followed by hexafluorobenzene (60 μL, 25 eq), the reaction solution isstirred for another 5 hours. The desired product is obtained by weakcation exchange chromatography (CM-Sepharose) followed by solid phaseextraction (Amberchrome CG300M) to desalt, and finally lyophilization.MALDI/TOF mass spectrum (m/z): calcd: 9975; found: 9976 (M+H).

Example 5: HeLa Cell Assay

The graph in FIG. 1 (see also Table 3) shows on the y axis “meanfluorescence intensity” of e-GFP protein green fluorescence emission asread from each individual cell by an Accuri C6 flow cytometer and summedby the processing software. The height of the green fluorescent signalbars indicate different intensities based on the level of exon skippingthat occurs in the nucleus of HeLa-654 cells, whether the cells aretreated or untreated with test compounds. The signal intensity or sizeof the bar also corresponds to the effectiveness of cellular uptake(that is, delivery into the cell). The x axis shows test compoundstested with a variety of different treatment regimens. The varioustreatment regimens include continuous treatment of HeLa-654 cells overtime or pulse-chase treatments where a test compound is incubated withcells and then after a period of time (3 hours) is washed away from thecells and fresh, test compound-free media is added.

Results:

The baseline activity of unconjugated PMO and untreated cells is shown.The peptide/PMO (PPMO) conjugates of the present disclosure all showgreater cellular uptake than: 1) unconjugated PMO, and 2) untreatedcells (which reveal the green fluorescent background signal). Thisdifference in fluorescence intensity between the peptide/PMO conjugatesof the present invention and the background or unconjugated-PMOindicates significant pharmacological activity of the stapledpeptide/PMO conjugates.

In each case, the PMO is of the following structure:

where the nucleobase sequence (comprising each occurrence of R² beingindependently selected from a nucleobase selected from A, G, C or T) is:GCT ATT ACC TTA ACC CAG (SEQ ID NO:9).

TABLE 3 Cellular Delivery of peptide-oligonucleotide-conjugates (FIG. 1)Mean FL1-H No Treatment 36,594.96 PMO 43,039.34 (i, i + 4) Hexa - PPMO 3123,401.08 (i, i + 4) Deca - PPMO 1 87,951.78 (i, i + 7) Deca - PPMO 4137,184.08

PPMO 3 refers to the following structure:

where the nucleobase sequence (comprising each occurrence of R² beingindependently selected from a nucleobase selected from A, G, C or T) is:GCT ATT ACC TTA ACC CAG (SEQ ID NO:9).

PPMO 4 refers to the following structure:

where the nucleobase sequence (comprising each occurrence of R² beingindependently selected from a nucleobase selected from A, G, C or T) is:GCT ATT ACC TTA ACC CAG (SEQ ID NO:9).

Example 6: In Vivo Protein and Exon Skipping

14 groups of 5 mice each (70 mice total; 65 mdx, 5 wt) were treated asdescribed below, and then analyzed for dystrophin protein and exonskipping.

Mice were given a 200 μL bolus of compound via tail vein injection, andwere then euthanized 8 days post-injection. Quadriceps, diaphragm, heartand brain tissues (individual structures) were collected, lysed, andanalyzed for dystrophin protein using traditional western blot, andanalyzed for exon skipping using RT-PCR and Caliper (see FIGS. 2-7).

Embodiments of compounds of the present disclosure were tested for theirability in vivo to effect mRNA and protein remodeling. In the event,these tested compounds showed activity in the mdx mouse, causingsubstantial increases in exon 23 skipping and dystrophin proteinexpression as shown by RT-PCR and western blot, respectively. Thisactivity is especially pronounced when measured against the backgroundof negative control experiments where the mdx mouse was dosed withsaline, a condition which produced essentially zero exon 23 skipping anddystrophin (FIGS. 4D, 5C, 5D, 6D, 7C, and 7D). When compared to the twopositive controls, wild type mice and mdx mice dosed with a known activecompound Ac-R6-Gly-M23D(+7-18) (PPMO 5), the tested compounds wereclearly active. In some cases the activity was greater than control; forexample, PPMO 11 (Table 4) produced much larger responses in exonskipping and dystrophin expression at lower doses than the positivecontrol compound (FIGS. 7B and 7D). Thus the tested compounds meet thecondition of eliciting positive pharmacological responses in the contextof a seven day in vivo screening assay.

Embodiments of compounds (PPMOs) of the present disclosure used in thesestudies have a structure according to Formula V:

wherein

the nucleobase sequence (comprising each occurrence of R² beingindependently selected from a nucleobase selected from A, G, C or T) is:5′-GGC CAA ACC TCG GCT TAC CTG AAA T-3′ (SEQ ID NO:14); and

R¹⁴ is as defined in Table 4.

Compounds of Table 4 were prepared according to Examples 1-4 above.

TABLE 4 Compound of Formula (V) R¹⁴ (* see Table 2) PPMO 5 Ac-R₆-Gly-(SEQ ID NO: 15) PPMO 6 4AD* PPMO 7 3AD* PPMO 8 4AC* PPMO 9 3AC* PPMO 104AE* PPMO 11 3AE*

Dystrophin Western Blot Protocol:

1. Tissue was removed from −80° C. and manually chopped into smallpieces, 400-800 μL of RIPA lysis buffer added depending on the tissuetype.

a. TA: 400 QC: 800 Heart: 400 μL

b. Lysis buffer: RIPA buffer (Pierce, cat#89901) and proteinaseinhibitor cocktail (Roche, cat#04693124001)

2. About ten 2.0 mm Zirconia Beads (Biospec, Cat#11079124zx) were addedto the tube, and the tissues were lysed with MagNA Lyser (Roche) asfollows:

a. 5,000 rpm, 30-40 s, cooled for 2 min on ice. The cycles were repeateduntil the tissues were completely lysed.

Alternatively to Zirconia Beads, metal beads (3-5 beads) were used, 3000rpm, 20 s per cycle (cooled on ice between cycles for 3-5 minutes oruntil cold to the touch).

3. Following lysis, samples were centrifuged at 12,000 rpm for 10 minand the supernatant transferred to a fresh tube, and a BCA assay wasperformed to quantify protein levels.

4. Gel Electrophoresis:

a. Marker was loaded (Invitrogen, cat# P/NLC5699) and 100 μg of proteinwas loaded per lane, and the samples were run on a 3-8% Tris Acetate gel(midi gel, Biorad, cat#345-0130) at 50 v, 5 min; 150 v, 1 hr and then200 v for 1.5 hr (71 kD marker was at the bottom of the gel).

5. Transfer:

Overnight wet transfer (16-18 hr) at 4° C., constant 100 mA (˜25 v),0.45 μm PVDF membrane (Biorad, cat#1620261).

6. The membrane was washed in TBST for 5 min, and blocked in 10% milk(Biorad, cat#170-6404) in TBST for 1-2 hr at room temperature (RT).

7. The PVDF membrane was rinsed with TBST 2-3 times, and then incubatedwith primary antibody in 5% BSA in PBS overnight at 4° C.

a. Dys2 (Novocastra, Leica Biosystem, cat#NCL-DYS2) 1:30

b. Dys1 (Novocastra, Leica Biosystem, cat#NCL-DYS1) 1:1000

c. Mandys8 (Sigma, cat#D8168) 1:3000.

8. The membrane was washed with TBST (10 min for each time, 3 times),HRP conjugated goat anti-mouse (BioRad, cat#1706516, 1:10000) secondaryantibody was added, incubated for 1 hr at RT, and then washed with TBST(10 min for each time, 3 times).

9. Clarity Western ECL substrate (Biorad) was added and incubated for 5min at RT, and then visualized with Chemidoc touch imaging system.

10. The membrane was stripped with 0.2N NaOH for 7 min, thenequilibrated in TBST for at least 10 min. The blot was blocked with 10%milk in TBST for 1 hr at RT.

11. α-actinin2 antibody (Abcam, rabbit, cat# ab68168, 1:15000) was addedand incubated for 1 hr at RT.

12. Washed with TBST (10 min for each time, 3 times) and then incubatedwith the HRP conjugated goat anti-rabbit (BioRad, cat#1706515, 1:10000)secondary antibody for 1 hr at RT.

13. The membrane was washed with TBST extensively and then imaged asdescribed above.

RNA Extraction Method for Mouse Tissues:

1. Whole tissue pieces (half quadricep, half heart, whole TA . . . )were homogenized with MagNA Lyser and metal beads at 3000 rpm, 20 percycle until the tissues were lysed well (samples were cooled on ice inbetween cycles for 3-5 minutes or until cold to the touch). 50-100 μL ofwhole lysate were transferred into a new 96 well plate and mixed withthe same volume of RA4 buffer (from GE RNAspin Kit), the lysate mixturewas then loaded into the 96 well RNAspin plate for the remainingpurification steps. Unused lysate (in RA1 buffer) was stored at −80° C.for one year according to the kit protocol.

2. Samples were centrifuged for 2 min at 5200 g. 500 μL RA3 was added toeach well of RNA binding plate, and the plate was centrifuged for 2 minat 5200 g. The flow through was discarded.

3. The membrane was washed by adding 500 μL RA2 to each well of RNAbinding plate, and centrifuged for 2 min at 5200 g.

4. 800 μL RA3 was added to each well and centrifuged for 2 min at 5200g.

5. 500 μL RA4 was added to each well and centrifuged for 10 min at 5200g.

6. The samples were eluted into a PCR plate (with conical wells) bypipetting 50 μL RNase free water into the bottom of each well, ensuringthe membrane was completely wetted. Samples were incubated for 2 min atRT and centrifuged at 5200 g for 3 min. If there was any liquidremaining in the well, the samples were incubated for an additional 2minutes at RT and centrifuged again at 5200 g for 3 min.

7. The RNA concentration was then measured on a Nanodrop 2000spectrophotometer.

RT-PCR Protocol:

Primers used for RT-PCR were as follows: Dystrophin Outer Forward:5′-CAATGTTTCTGGATGCAGACTTTGTGG-3′; (SEQ ID NO: 10)Dystrophin Outer Reverse: 5′-GTTCAGCTTCACTCTTTATCTTCTGCC-3′;(SEQ ID NO: 11) Dystrophin Inner Forward: 5′-CACATCTTTGATGGTGTGAGG-3′;(SEQ ID NO: 12) and Dystrophin Inner Reverse:5′-CAACTTCAGCCATCCATTTCTG-3′. (SEQ ID NO: 13)

25 μL reactions (Table 5) were prepared for RT-PCR and primaryamplification. RT-PCR program was performed according to Table 6.

TABLE 5 RT-PCR Reaction Mixture 2x Reaction mix 12.5 μL Dys innerForward Primer (10 μM) 0.75 μL Dys inner Reverse Primer (10 μM) 0.75 μLSuperscript III Platinum Taq mix 1 μL Template RNA (RNA sample) 3 μLWater to 25 μL total volume 7 μL

TABLE 6 RT-PCR and Primary Amplification Program Temperature TimeReverse Transcription 55° C. 30 minutes RT Inactivation 94° C. 2 minutesDenaturing 94° C. 45 seconds 45 Cycles Annealing 59° C. 45 secondsExtention 68° C. 1 minute  4° C. ∞

Caliper Protocol:

After RT-PCR was completed, 25 uL of PCR buffer was added and the total50 uL of reaction mixture was transferred into a Caliper plate. CaliperLabChip bioanalysis was performed based on the manufacturers'recommended protocol. The PCR product from full-length dystrophintranscript is 445 bps, and 232 bps from exon 23-skipped mRNA.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties. Unless otherwise defined, alltechnical and scientific terms used herein are accorded the meaningcommonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A peptide-oligonucleotide-conjugate of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: A′ is selectedfrom —NHCH₂C(O)NH₂, —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

wherein R⁵ is —C(O)(O-alkyl)_(x)-OH, wherein x is 3-10 and each alkylgroup is independently at each occurrence C₂₋₆-alkyl, or R⁵ is selectedfrom —C(O)C₁₋₆ alkyl, trityl, monomethoxytrityl, —(C₁₋₆-alkyl)R⁶, —(C₁₋₆heteroalkyl)-R⁶, aryl-R⁶, heteroaryl-R⁶, —C(O)O—(C₁₋₆ alkyl)-R⁶,—C(O)O-aryl-R⁶, —C(O)O— heteroaryl-R⁶ and

wherein R⁶ is selected from OH, SH, and NH₂, or R⁶ is O, S, or NH,covalently linked to a solid support; each R¹ is independently selectedfrom OH and —NR³R⁴, wherein each R³ and R⁴ are independently at eachoccurrence —C₁₋₆ alkyl; each R² is independently selected from H, anucleobase, and a nucleobase functionalized with a chemicalprotecting-group, wherein the nucleobase independently at eachoccurrence comprises a C₃₋₆ heterocyclic ring selected from pyridine,pyrimidine, triazinane, purine, and deaza-purine; z is 8-40; and E′ isselected from H, —C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, benzoyl, stearoyl,trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

wherein Q is —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—, R⁷ is—(CH₂)₂OC(O)N(R⁸)₂, wherein R⁸ is —(CH₂)₆NHC(═NH)NH₂, and R¹¹ isselected from OH and —NR³R⁴, wherein L is covalently linked by an amidebond to the carboxy-terminus of J, and L is selected from—NH(CH₂)₁₋₆C(O)—, —NH(CH₂)₁₋₆C(O)NH(CH₂)₁₋₆C(O)—, and

t is 4-9; each J is independently at each occurrence selected from anamino acid of the structure

wherein: r and q are each independently 0, 1, 2, 3, or 4; and each R⁹ isindependently at each occurrence selected from H, an amino acidside-chain, and an amino acid side-chain functionalized with a chemicalprotecting-group, wherein two or more amino acid side-chain groups of R⁹independently at each occurrence comprise a sulfur, wherein two of thesulfur atoms, together with the atoms to which they are attached, formthe structure

wherein d is 0 or 1, and M is selected from:

wherein each R¹⁰ is independently at each occurrence H or a halogen; andG is covalently linked to the amino-terminus of J, and G is selectedfrom H, —C(O)C₁₋₆ alkyl, benzoyl, and stearoyl, and wherein at least oneof the following conditions is true: 1) A′ is

2) E′ is

or 3) E′ is


2. The peptide-oligonucleotide-conjugate of claim 1, or apharmaceutically acceptable salt thereof, wherein E′ is selected from H,—C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, benzoyl, stearoyl, trityl,monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and


3. The peptide-oligonucleotide-conjugate of claim 1, or apharmaceutically acceptable salt thereof, wherein A′ is


4. The peptide-oligonucleotide-conjugate of any one of claim 1, or apharmaceutically acceptable salt thereof, wherein A′ is selected from—N(C₁₋₆-alkyl)CH₂C(O)NH₂,


5. The peptide-oligonucleotide-conjugate of any one of claims 1-4, or apharmaceutically acceptable salt thereof, wherein E′ is selected from H,—C(O)CH₃, trityl, 4-methoxytrityl, benzoyl, stearoyl, and


6. The peptide-oligonucleotide-conjugate of any one of claims 1-5, or apharmaceutically acceptable salt thereof, wherein A′ is selected from—N(C₁₋₆-alkyl)CH₂C(O)NH₂,


7. The peptide-oligonucleotide-conjugate of claim 1, or apharmaceutically acceptable salt thereof, wherein A′ is

and E′ is selected from H, —C(O)CH₃, trityl, 4-methoxytrityl, benzoyl,and stearoyl.
 8. The peptide-oligonucleotide-conjugate of claim 1, or apharmaceutically acceptable salt thereof, wherein thepeptide-oligonucleotide-conjugate of Formula I is apeptide-oligonucleotide-conjugate selected from:


9. The peptide-oligonucleotide-conjugate of claim 1 or 8, or apharmaceutically acceptable salt thereof, wherein thepeptide-oligonucleotide-conjugate is of the formula (Ia) and R⁵ is—C(O)(O—CH₂CH₂)₃OH.
 10. The peptide-oligonucleotide-conjugate of claim 1or 8, or a pharmaceutically acceptable salt thereof, wherein thepeptide-oligonucleotide-conjugate is of the formula (Ib) and E′ isselected from H, C₁₋₆ alkyl, —C(O)CH₃, benzoyl, and stearoyl.
 11. Thepeptide-oligonucleotide-conjugate of any one of claims 1-10, or apharmaceutically acceptable salt thereof, wherein each R¹⁰ is fluorine.12. The peptide-oligonucleotide-conjugate of any one of claims 1-11, ora pharmaceutically acceptable salt thereof, wherein M is


13. The peptide-oligonucleotide-conjugate of any one of claims 1-12, ora pharmaceutically acceptable salt thereof, wherein J is independentlyselected from cysteine and arginine.
 14. Thepeptide-oligonucleotide-conjugate of any one of claims 1-13, or apharmaceutically acceptable salt thereof, wherein two J groups arecysteine.
 15. The peptide-oligonucleotide-conjugate of any one of claims1-14, or a pharmaceutically acceptable salt thereof, wherein z is 8-25.16. The peptide-oligonucleotide-conjugate of any one of claims 1-15, ora pharmaceutically acceptable salt thereof, wherein each R¹ is N(CH₃)₂.17. The peptide-oligonucleotide-conjugate of any one of claims 1-16, ora pharmaceutically acceptable salt thereof, wherein each R² is anucleobase independently at each occurrence selected from adenine,guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.18. The peptide-oligonucleotide-conjugate of any one of claims 1-17, ora pharmaceutically acceptable salt thereof, wherein L is selected from—NH(CH₂)₁₋₆C(O)—, —NH(CH₂)₅C(O)NH(CH₂)₂C(O)—, and


19. The peptide-oligonucleotide-conjugate of any one of claims 1-18, ora pharmaceutically acceptable salt thereof, wherein L is selected fromglycine and


20. The peptide-oligonucleotide-conjugate of any one of claims 1-19, ora pharmaceutically acceptable salt thereof, wherein L is glycine. 21.The peptide-oligonucleotide-conjugate of any one of claims 1-20, or apharmaceutically acceptable salt thereof, wherein G is selected from H,C(O)CH₃, benzoyl, and stearoyl.
 22. Thepeptide-oligonucleotide-conjugate of any one of claims 1-21, or apharmaceutically acceptable salt thereof, wherein G is H or —C(O)CH₃.23. The peptide-oligonucleotide-conjugate of any one of claims 1-22, ora pharmaceutically acceptable salt thereof, wherein G is —C(O)CH₃. 24.The peptide-oligonucleotide-conjugate of claim 1-23, or apharmaceutically acceptable salt thereof, wherein d is
 1. 25. Thepeptide-oligonucleotide-conjugate of any one of claims 1-23, or apharmaceutically acceptable salt thereof, wherein d is
 0. 26. Thepeptide-oligonucleotide-conjugate of any one of claims 1-24, or apharmaceutically acceptable salt thereof, wherein

is selected from:


27. The peptide-oligonucleotide-conjugate of any one of claims 1-24 and26, or a pharmaceutically acceptable salt thereof, wherein thepeptide-oligonucleotide-conjugate is selected from:

wherein G is selected from H and —C(O)CH₃, and E′ is selected from H and—C(O)CH₃.
 28. The peptide-oligonucleotide-conjugate of claim 27, whereinG is H.
 29. The peptide-oligonucleotide-conjugate of claim 27, wherein Gis —C(O)CH₃.
 30. The peptide-oligonucleotide-conjugate of claim 27,wherein E′ is H.
 31. The peptide-oligonucleotide-conjugate of claim 27,wherein E′ is —C(O)CH₃.
 32. The peptide-oligonucleotide-conjugate ofclaim 27, wherein E′ and G are —C(O)CH₃.
 33. Thepeptide-oligonucleotide-conjugate of claim 27, wherein G is —C(O)CH₃ andE′ is H.